1. science
2. ecology

Notes
in Daphnia: An improved two-compartment
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LI, W. K. W., H. E. GLOVER, AND I. MORRIS. 1980.
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Plenum.
PEIRSON, W. M. 1983. Utilization
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Biophys. 21: 16 l-l 83.
RAYMONT,
J. E. G., R. T. STRINIVASAGAM, AND J. K.
B. RAYMONT. 1969. Biochemical studies on marine zooplankton. 7. Observations on certain deepsea zooplankton. Int. Rev. Gesamten Hydrobiol.
54: 357-365.
RIVKIN, R. B. 1989. Influence of irradiance and spectral quality on the carbon metabolism of phytoplankton. 1. Photosynthesis,
chemical composi-
Lmnol.
Oceanogr., 36(4), 199 1, 807-8 14
0 199 1, by the American
Society of Limnology
and Oceanography,
807
tion and growth. Mar. Ecol. Prog. Ser. 55: 291304.
ROBERTS, R. B., D. B. COWIE, P. H. ABELSON, E. T.
BOLTON, AND R. J. BRII-TEN. 1955. Studies on
the biosynthesis in Escherichia coli. Publ. Carnegie
Inst. 607. 521 p.
ROMAN, M. R. 1984. Utilization
of detritus by the
copepod Acartia tonsa. Limnol. Oceanogr. 29: 949959.
SMITH, R. E. H., AND R. J. GEIDER. 1985. Kinetics of
intracellular carbon allocation in a marine diatom.
J. Exp. Mar. Biol. Ecol. 93: 19 l-210.
SMITH, S. L., AND B. K. HALL. 1980. Transfer of
radioactive carbon within the copepod Remora
longicornis. Mar. Biol. 55: 277-286.
SMUCKER, R. A., AND R. DAWSON. 1986. Products of
photosynthesis by marine phytoplankton:
Chitin
in TCA “protein”
precipitates. J. Exp. Mar. Biol.
Ecol. 104: 143-152.
TENORE, K. R., AND L. GUIDI. 1984. Carbon-14 net
incorporation
does not accurately estimate the
weight-specific growth rate of the polychaete Capitella capitata. Mar. Biol. 79: 101-107.
WALNE, P. R. 1973. Growth rates and nitrogen and
carbohydrate contents of juvenile clams, Saxidomus giganteus, fed three species of algae. J. Fish.
Res. Bd. Can. 30: 1825-l 830.
Submitted: 27 June 1989
Accepted: 6 November 1990
Revised: 14 January 1991
Inc.
Carbon, nitrogen, and phosphorus content of
freshwater zooplankton
Abstract-The amounts of C, N, and P in relation to dry weight were measured in natural
populations of crustacean zooplankton
from a
humic lake. The elemental composition within a
given species showed little seasonal variation, and
experimental starvation or feeding did not cause
any significant changes in the P content. C constituted 48+ 1% of dry weight with only minor
variation
among species. The mean C: N: P
atomic ratios reflected large interspecific differences in P and N content and ranged from 2 12 :
39: 1 in Acanthodiaptomus denticornis to 85 :
14 : 1 in Daphnia longispina. Comparisons with
Acknowledgments
This work was financed by the research program on
eutrophication
of inland waters, supported by the Royal Norwegian Council for Scientific and Industrial Research.
We thank Anne Lyche and two anonymous reviewers for comments on earlier drafts of this paper.
published measurements indicate that this pattern is quite general and probably constitutional.
These findings suggest that the species composition of the zooplankton community can have
strong influence on the N : P ratio of recycled nutrients and thereby affect resource competition
between phytoplankton
species.
Herbivorous
zooplankton
influence the
proliferation
of planktonic algae and bacteria through the simultaneous effects of
grazing and nutrient recycling. As pointed
out by Sterner (1989), zooplankton act not
only as predators in a classical sense, but
also have an indirect effect on resource competition between algal species and bacteria.
The major forms of dissolved P and N released from zooplankton are easily accessible to phytoplankton
and may be a major
808
Notes
nutrient supply to the primary producers at
certain times (Peters and Rigler 1973; Lehman 1980, 1984).
Olsen and 0stgaard (1985) proposed a
model wherein nutrient recycling was given
implicitly by the balance between ingestion
and utilization into somatic growth and reproduction. In a following paper, Olsen et
al. (1986) supported this concept with results from field experiments,
showing a
strong dependence of the P release rate of
Daphnia on the P : C ratio of the food particles. Sterner (1989) extended these ideas
to a conceptual model in which grazers ingest food with highly variable C : N : P ratios
while assimilating
a relatively
constant
C : N : P ratio, releasing the difference between the two. This inference is in accord
with the observation of Lehman (1984) that
the egested portion of ingested food is generally more variable than the assimilated
portion. Under these assumptions the zooplankton would drain a constant C : N : P
proportion from food particles, resulting in
a progressively diverging N : P ratio in the
recycled nutrients which would be expected
to have a strong influence on the competition between phytoplankton
species (e.g.
Kilham and Kilham 1984).
Sterner (1989), admitting the lack of relevant data from freshwater localities, based
the assumption of constant zooplankton
C: N: P ratios mainly on extrapolations
from the marine literature which indicates
that N content is less variable in marine
zooplankton than in phytoplankton. We here
present measurements of C, N, and P content in six species of freshwater zooplankton- both in the field and under experimental conditions-as
part of an integrated
study of the zooplankton
community
in
Kjelsasputten, a small, acid, brown-water
seepage lake located in a forest area near
Oslo.
During the period of investigation,
particulate C averaged 0.43 mg C liter-’ (Fig.
I), with detritus probably making up 7590% (Hessen et al. 1990). Concentrations of
particulate P and N were low, with the seston P : C ratio more variable than the N : C
ratio (Fig. 1). The zooplankton community
was dominated by the cladocerans Daphnia
longispina, Holopedium gibberum, Diapha-
9
0
1.6
:,
1.2
E
h’
0.6
F
0.4
I
May
-8
Jun
JUI
Aw
Sep
act
Fig. 1. Seasonal variation in seston composition in
Kjelshputten,
1986 [pooled samples from epilimnion
(O-3 m); see Hessen 19891. Lower panel-particulate
C; middle panel-N
: C ratio (by wt); upper panel P: C ratio (by wt).
nosoma brachyurum, and Bosmina longispina and the Calanoid copepods Acanthodiaptomus denticornis and Heterocope
saliens; rotifers and cyclopoid copepods
made up < 5% of the zooplankton biomass.
Further details on C cycling, community
structure, and seasonal succession can be
found elsewhere (Hessen 1989; Hessen et
al. 1990).
Field samples of zooplankton were taken
by vertical (45-pm mesh size) net hauls at
about biweekly intervals throughout
the
growing season from May to November
1986. Live animals were brought in lake
water to the laboratory, collected on nylon
screens, and immediately frozen (- 20°C).
Before analysis, frozen animals were directly sorted by species and developmental
stage while they thawed under a dissecting
microscope. Care was taken to pick animals
immediately upon thawing to prevent leakage of body fluids. Each sample consisted
of 3-30 adults of similar size, giving an average sample size (rt 1 SD) of 157 + 8 5 pg
dry weight (DW).
Three parallel laboratory
experiments
were performed in a 17°C constant temperature room under an artificial 12 : 12 L/D
Notes
cycle. In each experiment a mixture of 2050 adults of D. longispina, H. gibberum, and
B. longispina were added to 5 liters of water.
The receiving natural lake water was given
the following treatments: food removal (filtration through Whatman GF/F glass-fiber
filters), inorganic P enrichment (addition of
10 pg Pod-P liter-‘), and food enrichment
(addition of exponentially growing algae and
bacteria).
At the end of the experiment, the animals
were harvested and 2-4 replicates analyzed
for dry weight and P content after the same
analytical procedure as the field samples,
except that animals were processed live after anesthesia with carbonated water. The
starvation experiment was terminated after
4 d when mortality increased rapidly, and
the other two experiments were terminated
after 2 weeks. Tests with 33P043- showed
that the phosphate addition was taken up
in < 1 h, with small particles (< 3 pm) containing 90% of the isotope. The inorganic P
addition represented an approximate
tripling of the particulate
P concentration
compared to the lake situation; the food addition amounted to more than a lo-fold increase in particulate P. From the final P
analyses, we calculate that the zooplankton
addition per se amounted to - 1 pg P liter- l.
The food enrichment consisted of algae from
a mixed culture of cryptomonad species at
the equivalent of 10 pg Chl a liter-l and
bacteria from a Pseudomonas strain isolated
from the same locality at a final concentration of - 5 X lo6 cells ml-l.
Specimens for C and N analysis were
placed in preweighed tin capsules; preweighed polycarbonate capsules were used
for the P analyses. Dry weights of the samples were measured on a Mettler ME30 microbalance after drying overnight at 60°C.
Five-ten
blank capsules were carried
through the procedure to correct for differences in air humidity and drift in the calibration of the weighing equipment. C and
N contents were measured on a Carlo-Erba
CHN 1106 elemental analyzer. P analyses
were performed in 7-ml scintillation
minivials. All capsules, vials, and screwcaps were
cleaned by soaking in antimony-molybdate
solution and rinsing twice in double-distilled water. Samples were first oxidized
809
overnight with 200 ~1 of H202 at room temperature to make the exoskeleton more hydrophilic and to avoid incomplete digestion
of animals floating on the surface. The samples were then digested in 2 ml of K&O8
solution (10 g liter-l) for 1 h at 120°C. If
we assume that 2 mol of K&O8 is needed
to oxidize an amount of organic C equivalent to 1 mol of CO*, the amount added
would be sufficient to digest about sixfold
the average sample dry weight (DW). Tests
with animals reared on 33P-labeled food
showed insignificant amounts of radioactivity in the exoskeleton residues after this digestion procedure.
Standards were prepared by adding O-50~1 quantities of a stock orthophosphate solution (100 mg P liter- ‘) to minivials containing the empty polycarbonate capsules
used as blanks in the weighing procedure.
Letting the vials stand uncapped overnight
at room temperature was sufficient to evaporate the added water. All standards were
carried through the same digestion procedure as the samples. The rest of the analysis
closely followed standard molybdate-blue
methods for analysis of orthophosphate. The
antimony-molybdate
complexing
reagent
and the ascorbic acid reductant were prepared to yield correct final concentration
after 200 ~1 was added to each solution to
give a nominal final sample volume of 2.6
ml. The absorbance at 880 nm in l-cm cuvettes was linear with added orthophosphate over the whole range of O-5 pg P
vial- l. As standards were prepared in terms
of absolute amounts per vial instead of concentrations, the results were unaffected by
any systematic water loss due to evaporation during digestion.
P content was generally more variable
than N and C contents in the field samples
of all zooplankton species (Fig. 2). The comparison circles in the lower panels of Fig. 2
show that there were more significant interspecific differences in P content than in
C and N contents. The species could tentatively be ranked, as done in Fig. 2, with
decreasing C and N content and increasing
P content as A. denticornis, H. saliens, B.
longispina, H. gibber-urn, D. brachyurum,
and D. longispina. One-way ANOVA indicated significant variation among species
Notes
810
% Carbon
% Phosphorus
% Nitrogen
8
I
10
I
I
Acanthodiaptomus
I
i
0.6
I
12
I
:
I
III
1.0
I
1.4
I
1.8
I
Ii
Heterocope
Bosmina
I
Holopedium
I
I::!
:
Diaphanosoma
I
:
i
.:
Ha-i
I
:::I
1::::
I
Daphnia
Fig. 2. Box-and-whisker
displays of distributions
of all field samples by zooplankton species and element
(C, N, and P as percent of dry wt). Boxes indicate medians and the middle two quartiles; whiskers indicate the
limits of the lO-90% percentiles. Box heights are proportional
to sample sizes. Lower panels show comparison
circles among species within an element; two distributions are considered different at a 95% C.L. if their circles
are disjunct or have an outside angle of intersection ~90”.
for all three elements (C: F5,96 = 4.93, P =
0.00 12; N: F5,94= 16.6, P= 0.0001; P: F5,101
= 83.1, P < 0.000 l), but tests by multiple
Scheffe comparisons showed that many of
the differences between adjacent species were
not significant at the 5% level.
In the laboratory experiments, the starvation treatment gave negligible egg production and rapidly increasing mortality in
D. longispina by the end of the experiment.
Phosphate addition alone did not change
the egg production rate in any species, al-
though animals fed with algae and bacteria
increased egg production by threefold-fivefold compared to the field situation. This
result makes it likely that secondary production at the time of the experiment was
limited by food quantity and not food quality in terms of P content. The results from
the experimental treatments (Fig. 3) did not
differ significantly from the field samples in
B. longispina (F3,26= 0.59, P = 0.63) and
D. longispina (F3,45= 1.65, P = 0.19), but
a significant difference was found in H. gib-
% Phosphorus of dry weight
0.5
I
I
1.0
I
I
1.5
I
I
0.5
I
I
1.0
I
I
1.5
I
0.5
1.0
1.5
2.0
- Food
+P
+ Food
Daphnia
Holopedium
Bosmina
Fig. 3. Box-and-whisker
displays of distributions of P contents in all samples from the laboratory
by zooplankton species and treatment. Details same as for Fig. 2.
experiments
Notes
811
Heterocope
E
.o,
I:: m
:: t
1
g
a%
6
1.5~-
I
Holopedium
1
I
I
Acanthodiaptomus
!
Daphnia
I
May
Jun
Jul
Aug
Sep
Ott
May
Jun
Jul
Fig. 4. Seasonal variation in P content in the zooplankton species investigated.
of replicates-0
(range of replicated measurements indicated by vertical bars).
berum (F3,27= 6.07, P = 0.0027). The most
significant effect on H. gibberum was due to
phosphate addition, which might be caused
by uptake of inorganic P by the microflora
attached to the gelatinous envelope of this
species (Hessen et al. 1990). Excluding this
treatment from the analysis showed no significant difference between the other treatments and the field observations (F2,23=
2.60, P = 0.096).
Both seston and zooplankton composition showed the highest variability in P content. There was a clear seasonal trend in
seston P content with low P : C in spring and
high P : C in autumn (Fig. 1). Most zooplankton species showed some temporal
variation (Fig. 4) that could not be explained by intraspecific variability
or analytical errors, but the lack of correspondence
among species makes it unlikely that this
variation is the result of a common response
to changes in the seston P : C ratio. At least
for the copepods, we suspect that some of
Aug
Sep
Single samples-O;
Ott
means
the temporal pattern is related to specific
life-history phenomena.
Seasonal changes in C and N content are
well documented in marine copepods from
temperate and boreal areas (see Bamstedt
1986). The most pronounced variation in
boreal species with univoltine life histories
seems to be connected with lipid allocation
strategies for surviving winter, so that one
would expect less variability
in freshwater
species that survive the winter as resting
stages. Baudouin and Ravera (1972) found
no evidence of a seasonal pattern in the
chemical composition of Daphnia hyalina
from oligotrophic
Lago di Monate. Behrendt (1990) found some temporal variability in zooplankton chemical composition in Grof3er Miiggelsee, but his results
were based on total zooplankton samples,
so the variation might be attributable
to
changes in species composition.
Lehman and Naumoski (1985) reported
that Daphnia pulex fed P-sufficient green
Notes
812
Table 1. Summary of published measurements of P content in Daphnia species. Values from Vijverberg and
Frank (1976) were recalculated assuming that total organic matter measured by chemical oxygen demand equals
dry weight minus 7% chitin and 10% ash.
Mean
D. pulex
D. pulex
D. pulex
D. hyalina
D. hyalina
D. rosea
D. galeata
1.53
1.25
1.53
1.12
1.66
1.80
1.11
SD
n
Reference
0.06
0.32
0.23
0.10
0.17
0.21
0.14
3
56
5
30
9
60
5
Birge and Juday 1922
Lehman 1980
Langeland et al. 1985
Baudouin and Ravera 1972
Vijverberg and Frank 1976
Peters and Rigler 1973
Langeland et al. 1985
algae had a higher P content than individuals reared on P-deficient algae. Their conclusion was based on changes in the log-log
regression of P per individual on individual
length, with varying P status in the food
algae. When comparing freshly molted individuals without eggs, the length-weight relationship in Daphnia is unaffected by food
conditions (Lynch 1989). Without this precaution, this allometry can be quite variable
and has often been used as an indicator of
the prevailing growth conditions (e.g. Duncan 1985). The results of Lehman and Naumoski (198 5) could therefore also be interpreted as a change in the length-weight
relationship
caused by improved growth
conditions, instead of a change in P content.
Such an interpretation
is supported by the
prevalence of noneggbearing females in their
low-P treatments (Lehman and Naumoski
1985, figure 1).
The N and C contents we found all fall
within the observed range in marine copepods as reviewed by BAmstedt ( 1986, tables
1.11 and 1.12), although a substantial number of our P measurements are above those
reported in marine copepods (Bamstedt
1986, table 1.13). On the other hand, the
mean P content in the two Calanoid copepods we considered (0.6 5 + 0.19% P of DW,
n = 33) is close to the marine species investigated (0.76&O. 18% P of DW, n = 9).
The close correspondence between the elemental ratios of the herbivorous-detrivorous A. denticornis, the carnivorous H. saliens, and their marine relatives suggests that
a comparatively
low P content might be a
common property of Calanoid copepods.
Observations of P content in several Daphnia species (Table 1) give a mean value of
1.43 +0.27% P of DW, which compares fa-
vorably with the mean value of our estimates for D. longispina (1.47 + 0.17% P of
DW). The consistently high P content across
species and growth conditions indicates that
it might be a generic property of Daphnia
SPPUnicellular
microplankton
generally exhibit a flexible storage strategy with a large
capacity for accumulating materials in excess of immediate demands for growth and
maintenance (e.g. accumulation
of polyphosphate under high P availability
or carbohydrate under nutrient limitation). On the
basis of the small amount of variation in
zooplankton elemental ratios that could be
attributed to changes in food conditions,
such capacities seem to be limited in the
species investigated. This result suggests that
for many purposes the elemental composition of metazoan zooplankton can be considered constant and species-specific. Fixed
elemental ratios put important constraints
on the food utilization
of zooplankton in
that intake of a given element in excess of
immediate demands must be disposed by
increased excretion or by reduced assimilation of certain food components. This view
is supported by the results of Lehman and
Naumoski (1985), who found large differences between rates of gross P assimilation
and P excretion for D. pulex fed algae with
high and low P content.
The predicted effect of food composition
on nutrient recycling can be illustrated by
partitioning
the N : C, P : C plane into two
regions demarcated by a straight line through
the C : N : P ratio of the grazer (Fig. 5). Food
items with C : N : P ratios in the upper region will have higher N : P ratios than that
of the grazer and therefore contain more N
than is required for balanced growth, im-
Notes
0.16
--
0.12
--
z
E
8
2.
.-0
EJ
0
z
0.002
0.004
0.006
0.008
0.010
P : C ratio (atoms)
Fig. 5. C : N : P ratios in lake seston (0) in relation
to the N : P ratios required for balanced growth (lines)
in Acanthodiaptomus denticornis and Daphnia longispina. A and D indicate the measured C : N : P ratios
of A. denticornis (212 : 39 : 1) and D. longispina (85 :
14: 1); R shows the Redfield ratio (106: 16: 1).
plying an increased N : P ratio in recycled
nutrients. Food items with C : N : P ratios
in the lower region will have lower N : P
ratios than that of the grazer and therefore
contain more P than is required for balanced growth, implying a decreased N : P
ratio in recycled nutrients. Figure 5 indicates that all the seston samples from the
lake contain surplus N compared to the requirements of D. longispina, and that all
except two samples contain surplus P compared to the requirements of A. denticornis.
We thus expect D. longispina to have a higher N : P ratio in released nutrients than A.
denticornis in the lake investigated.
A general tendency for higher N : P ratio
in recycled nutrients
from zooplankton
communities dominated by daphnids than
from copepod-dominated
communities can
explain the results of Elser et al. (1988), who
found distinct shifts between N- and P-limited phytoplankton
growth accompanying
changes in zooplankton community structure. Both experimental and field studies
showed that N limitation prevailed in communities dominated by small species (mainly cyclopoid copepods), while dominance of
large cladocerans (mainly daphnids) led to
P limitation.
This pattern is exactly what would be pre-
813
dicted if cyclopoids, as suggested by the results of Kahn and Siddiqui (197 l), are assumed to have the same kind of efficient P
utilization as Calanoid copepods. Resource
competition theory predicts that the N : P
ratio of the nutrient supply can be a strong
determinant for the species composition of
the phytoplankton community (Kilham and
Kilham 1984). It leads us to suggest that, in
addition to the effects of selective grazing,
zooplankton
community
structure could
have an indirect selective force on the species composition
of the phytoplankton
community through the N : P ratio of the
recycled nutrients. As cyanobacteria seem
to be favored by low N: P supply ratios
(Smith 1983), this might offer an additional
explanation of the ability of daphnids to
suppress cyanobacterial blooms.
Tom Andersen
Department of Biology
University of Oslo
P.O. Box 1069 Blindern
N-03 16 Oslo 3, Norway
Dag 0. Hessen
Department of Biology
University of Oslo
P.O. Box 1050 Blindern
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Submitted: 1 April 1988
Accepted: 18 March 1991
Revised: 15 April 1991
Inc.
An improved fluorescence method for the determination of
nanomolar concentrations of ammonium in natural waters
Abstract -An improved fluorescence method
is described for measuring nanomolar concentrations of NH,+ in natural waters. This method
is based on the conversion of NH,+ to NH, and
subsequent diffusion of NH, across a microporous hydrophobic Teflon membrane into a flowing stream of o-phthaldialdehyde
reagent to produce a fluorescent adduct. The product is detected
fluorometrically
with a lower detection limit of
better than 1.5 nM. Up to 30 determinations h-l
can be made. The method works well in freshwater or salt water. Field tests of the method in
the Dry Tortugas and Gulf Stream gave NH,+
concentrations that ranged from 18.0 nM in Gulf
Stream waters to 2,254.7 nM in interstitial waters
Acknowledgments
This work was supported by the National Science
Foundation through grant OCE 86-20249.
I thank Capt. Millender
and the crew of the RV
Bellows for assistance during the field tests and A. Holloway for preparing the figures. I thank M. A. Brzezinski and J. H. Sharp for reviews.
from coralline reef sands. The method can be
used to measure near real-time NH,+ concentrations in situations where it was previously difficult or impossible.
The measurement of NH4+ in natural waters is key to understanding several aspects
of the aquatic nitrogen cycle (Carpenter and
Capone 1983). Often this measurement has
been hampered by detection limits or the
time involved in determining
nanomolar
concentrations of NH4+. Brzezinski (1987,
1988) has developed and applied a method
for calorimetric
determination
of nanomolar concentrations of NH,+ using a solvent
extraction technique. Although his method
has a detection limit of 3.5 nM, the procedure of extraction and analysis is slow and
labor intensive, allowing only 30 samples
to be processed in an 8-h day (Brzezinski